This proposal is a continuation of ongoing studies into the mechanisms of antibiotic-resistance gene structure, expression, and transfer in Bacteroides spp. These organisms are important components of the normal flora of the extremes of the gastrointestinal tract and are substantially phylogenetically diverged from other common human bacterial pathogens. Bacteroides fragilis, in particular, is an important pathogen with a propensity for abscess formation. Antimicrobial resistance has developed to many B-lactams and other drugs used to treat these organisms. The long term goal of the project is to elucidate the novel mechanisms that regulate expression of antibiotic resistance and virulence in these organisms and the systems that contribute to the dissemination of resistance genes. Dr. Smith's prior work identified the hybrid promoter structure of B-lactamase gene, cepA, involving IS1224 in strains expressing high and low levels of B-lactamase and has defined the common occurrence of apparent IS elements just upstream of antibiotic-resistance gene promoters. He has also cloned and characterized two other B-lactamase genes from Bacteroides spp. and characterized the structure of the mobilizable cefoxitin-resistance transposon element Tn4555, its Mob region, and its apparent oriT site, which is required in cis for transfer by conjugation. Tn4555 appears to differ from the previously reported NBU1 element. The specific aims of the current proposal are first to determine the mechanisms activating expression of the antibiotic resistance genes using the B. fragilis cepA gene as a model. Under this aim Dr. Smith will delineate the cepA::IS1224 hybrid promoter, perform structural analysis on IS-linked promoters, and define the mechanisms involved in IS1224 transposition. Under the second aim Dr. Smith proposes to define the mechanisms responsible for the dissemination of broad-spectrum B-lactam resistance in Bacteroides. For this he will focus on Tn4555 and the requirements for its mobilization for conjugation and for its transposition.